CN105832682A - Method for preparing honeycomb silk fibroin porous microsphere sustained drug release vector - Google Patents
Method for preparing honeycomb silk fibroin porous microsphere sustained drug release vector Download PDFInfo
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- CN105832682A CN105832682A CN201610369157.0A CN201610369157A CN105832682A CN 105832682 A CN105832682 A CN 105832682A CN 201610369157 A CN201610369157 A CN 201610369157A CN 105832682 A CN105832682 A CN 105832682A
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- silk fibroin
- fibroin
- porous microsphere
- silk
- drug release
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- 108010022355 Fibroins Proteins 0.000 title claims abstract description 107
- 239000004005 microsphere Substances 0.000 title claims abstract description 49
- 238000000034 method Methods 0.000 title claims abstract description 28
- 238000013269 sustained drug release Methods 0.000 title abstract description 8
- 239000003814 drug Substances 0.000 claims abstract description 76
- 229940079593 drug Drugs 0.000 claims abstract description 40
- VSIIXMUUUJUKCM-UHFFFAOYSA-D pentacalcium;fluoride;triphosphate Chemical compound [F-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O VSIIXMUUUJUKCM-UHFFFAOYSA-D 0.000 claims abstract description 17
- 229910052586 apatite Inorganic materials 0.000 claims abstract description 16
- 239000000725 suspension Substances 0.000 claims abstract description 14
- 239000010839 body fluid Substances 0.000 claims abstract description 13
- 210000001124 body fluid Anatomy 0.000 claims abstract description 13
- 239000007864 aqueous solution Substances 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims abstract 2
- 239000000243 solution Substances 0.000 claims description 21
- 230000001413 cellular effect Effects 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 16
- 239000012890 simulated body fluid Substances 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000004088 simulation Methods 0.000 claims description 10
- 241000255789 Bombyx mori Species 0.000 claims description 9
- 239000008367 deionised water Substances 0.000 claims description 8
- 229910021641 deionized water Inorganic materials 0.000 claims description 8
- 238000004108 freeze drying Methods 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 230000003578 releasing effect Effects 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000001914 filtration Methods 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 238000011275 oncology therapy Methods 0.000 claims description 5
- 239000012460 protein solution Substances 0.000 claims description 5
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 239000006185 dispersion Substances 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- 241000255794 Bombyx mandarina Species 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 2
- 239000007832 Na2SO4 Substances 0.000 claims description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 2
- 229920001872 Spider silk Polymers 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 2
- 210000004907 gland Anatomy 0.000 claims description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 2
- 108010064995 silkworm fibroin Proteins 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 16
- 238000013268 sustained release Methods 0.000 abstract description 14
- 239000012730 sustained-release form Substances 0.000 abstract description 14
- 238000011068 loading method Methods 0.000 abstract description 5
- 231100000331 toxic Toxicity 0.000 abstract description 5
- 230000002588 toxic effect Effects 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 3
- 102000004190 Enzymes Human genes 0.000 abstract description 3
- 239000011148 porous material Substances 0.000 abstract description 2
- 238000000502 dialysis Methods 0.000 abstract 1
- 238000001035 drying Methods 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- 238000001132 ultrasonic dispersion Methods 0.000 abstract 1
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 29
- 229940009456 adriamycin Drugs 0.000 description 14
- 210000004027 cell Anatomy 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 7
- 230000033558 biomineral tissue development Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 229910052500 inorganic mineral Inorganic materials 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000011707 mineral Substances 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 210000004881 tumor cell Anatomy 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 210000000988 bone and bone Anatomy 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 3
- 230000006911 nucleation Effects 0.000 description 3
- 238000010899 nucleation Methods 0.000 description 3
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000001338 self-assembly Methods 0.000 description 3
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000002045 lasting effect Effects 0.000 description 2
- 239000011806 microball Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000005204 segregation Methods 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 1
- 241000255978 Antheraea pernyi Species 0.000 description 1
- 201000009030 Carcinoma Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000019738 Limestone Nutrition 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical group [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 101710089165 Protein white Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 239000011805 ball Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 210000001564 haversian system Anatomy 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000006028 limestone Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002077 nanosphere Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 230000001699 photocatalysis Effects 0.000 description 1
- 238000007146 photocatalysis Methods 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
- A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
- A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a method for preparing a honeycomb silk fibroin porous microsphere sustained drug release vector. Fibrous fibroin formed after cocoon degumming is dissolved, filtered, dialyzed and concentrated to obtain silk fibroin aqueous solution; the silk fibroin aqueous solution is subject to ultrasonic dispersion, placed in a dialysis bag and placed in simulated human body fluids of different times concentrations to be soaked, the silk fibroin aqueous solution is processed in a uniform shaking table, the simulated human body fluids are replaced at time intervals to obtain microsphere suspension; and the microsphere suspension is washed many times and undergoes centrifugal separation and drying to obtain apatite/silk fibroin microsphere powder as the sustained drug release vector. Apatite microspheres of a certain size and pore diameters can be formed on the surface of silk fibroin, the dispersity, drug loading performance and sustained release performance are excellent, the honeycomb silk fibroin porous microsphere sustained drug release vector has no toxic or side effect on normal cells, the biocompatibility of the drug vector is improved remarkably, and the honeycomb silk fibroin porous microsphere sustained drug release vector has wide application prospects in the fields such as sustained drug release, tissue engineering and enzyme engineering.
Description
Technical field
The invention discloses the preparation method of a kind of cellular silk fibroin porous microsphere drug slow-released carrier, belong to
Biological medicine Material Field.
Background technology
Silk fiber is one of native protein of utilizing the earliest of the mankind, the native protein being mainly made up of fibroin albumen
Matter fiber is a kind of degradable biological macromolecular material, has excellent biocompatibility, mechanical property and low
Immunogenicity, is the ideal material for preparing biomaterial.In recent years, cure at biology along with fibroin material
The high speed development of medicine technology, is widely used to health products, biological medicine, organizational project, medicament slow release etc. all
Multi-field..
The effect of slow releasing carrier of medication is to provide lasting medicine to deliver medicine-feeding part (such as: tumor tissues),
Medicament slow release through long-time (such as, a couple of days or several weeks) is the convenience that patient provides administration, and subtracts
Lack therapeutic frequency, reduced nurse fees use, decrease rapid delivery of pharmaceuticals to other health tissues of health simultaneously
The side effect caused.In drug controlled release system, pharmaceutical carrier plays critical effect.Due to fibroin albumen
Solution is easy to sex change under the conditions of changing thing, changing, and therefore by changing the pH value in silk fibroin solution, adds
The methods such as alcoholic solution both can obtain fibroin microsphere.But, current fibroin albumen grinds for drug release carrier
Studying carefully the most at the early-stage, the fibroin microsphere that great majority research obtains has smooth surface, and at water solution system
, therefore there is medicine carrying single in middle extremely unstable, biological stability is poor, and drugloading rate is few, and sustained drug release effect is not
The shortcoming such as good, this most all limits the application in medicament slow release field of the fibroin albumen microballoon.Such as Shen
Please be on November 11st, 2011 day, in the Chinese patent of Publication No. CN201110357105.9, disclose
A kind of preparation method of silk fibroin nanosphere.Prepared microballoon table is can be seen that from the accompanying drawing 1 of this patent
Face becomes smooth spherical, and poor dispersion easily attracts each other and settles, can significantly reduce medicine
Embedding and releasing effect, therefore can not meet the needs of medicament slow release and production.
In sum, there is presently no a kind of operation simple, medicine carrying effect and the good fibroin albumen of releasing effect
Porous microsphere preparation method.Therefore, exploitation has porous pattern, and drugloading rate is high, has sustained drug release effect
Fibroin albumen microballoon has been very urgent.
Hydroxyapatite is to constitute natural bone and the main inorganic composition of tooth, has good biocompatibility.
Due to the production cost that it is relatively low, good water solubility, bigger specific surface area, and self have amphipathic etc.
Feature, is being applied to research field the most widely.The minimum microstructure of animal body inner bone tissues is for receiving
The brilliant apatite ordered fabrication structure on collagen fabric of rice, collagenous fibres structure in a regular array is phosphorus
The offer forming core site of lime stone also controls its orientation of growth, forms length and is about 30-50nm, and width is 10-
The osteon of 30nm.Therefore from bionical angle, this characteristic is utilized, by bionical mineralization method,
Utilize fibroin albumen to regulate and control nucleation and the growth of mineral, prepare the fibroin albumen microballoon of porous, can be used for medicine
The application of thing sustained release.Use fibroin albumen to regulate and control hydroxyapatite also to rarely have in order to the application in medicament slow release field
Research.For expensive cancer therapy drug or gene repair medicine, need high drug load and sustained release prolongation of effect, to reach
To optimal medicine carrying and releasing effect.
Summary of the invention
Overcoming and existing prepare above-mentioned deficiency present in fibroin albumen Microspheres Technique, the present invention proposes a kind of honeycomb
The preparation method of shape silk fibroin porous microsphere drug slow-released carrier.
Situation based on background technology, the present invention feature of protein mutability, induce hydroxyl under given conditions
Base apatite forms porous microsphere in fibroin protein surface-assembled, and this fibroin porous microsphere has good dispersion
Property, higher specific surface area, good biocompatibility, lasting slow release effect, the most thorough
Solve above problem.
Technical scheme is as follows:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain silk
The fibroin aqueous solution；
(2) the silk fibroin protein solution concentration in step (1) is adjusted to 0.1～50mg/mL and ultrasonic carries out
Dispersion, loads in the human simulation body fluid being placed in different times concentration in bag filter and soaks, and solution temperature is 37 DEG C,
At the uniform velocity shaking table processes；
(3) shaking table processes the time is 1-14 days, the human simulation body fluid in every 24 hours exchonge step (2),
Obtain microballoon suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing 3～5 times, centrifugal
Separate, be dried 5-48 hour at freeze-drying or 60 DEG C, through mimic biology mineralization process, at fibroin albumen base
In situ nucleation have cellular porous microsphere structure through being self-assembly of in matter, particle diameter is 0.2～5 μm, micro-
With honeycomb structure on ball, the diameter of aperture is about 20-100nm, as slow releasing carrier of medication.
The present invention utilizes simulated body fluid biomimetic mineralization method, by the functional functional group in fibroin albumen peptide chain
(such as carboxyl, amino etc.) induce apatite mineral in fibroin albumen surface fast nucleation and growth so that
Apatite carries out orderly self assembly and deposition on fibroin albumen surface, obtains the silk with cellular porous outer surface
Element/apatite microballoon, its electric charge is electronegative.
Fibroin raw material in described step (1) uses bombyx mori silk fibroin, silk gland protein, wild silkworm fibroin, spider silk protein
White or recombinant fibroin, but it is not limited to this.
One times of concentration of described human simulation body fluid uses mode to prepare: add in order in 800ml deionized water
Enter NaCl:7.996g, NaHCO3: 0.350g, KCl:0.224g, K2HPO43H2O:0.228g,
MgCl2·6H2O:0.305g, CaCl2: 0.278g, Na2SO4: 0.071g, then with the three of 50mmol/L
The pH value of simulated body fluid is adjusted to 7.40 by hydroxymethyl aminomethane/hydrochloride buffer, is settled to 1000ml, i.e. joins
Simulated body fluid used in cost experiment.
In described step (2), the proportioning of simulated body fluid includes but not limited to described simulated body fluid proportioning, can be upper
The compound method stating simulated body fluid carries out suitable amendment: the amount of chemical reagent can change, part chemical reagent
Can be replaced；In like manner, it is possible to (simulated body fluid ion concentration is multiplied by accordingly to configure the simulated body fluid of 1.5-5 times
Multiple) for the application of the present invention.
Described size of pharmaceutical particles is less than the cellular pore size in apatite/fibroin albumen microballoon.
Described medicine includes the cancer therapy drugs such as ADMh but is not limited to cancer therapy drug it can also be used to other medicine
The bag of thing carries.
The present invention is shown by the human body cell biocompatibility experiment of embodiment, non-medicine carrying silk fibroin porous
Microballoon has good biocompatibility.
The human body cell biocompatibility experiment of the present invention by the mass ratio of medicine with fibroin albumen microballoon is specifically
(1～50): drug solution is well-dispersed in fibroin albumen microspheres solution by 100, shake on shaking table,
37 DEG C of temperature are bathed 12 hours, obtain wrapping the fibroin porous microsphere being loaded with medicine.
The principle of the present invention is, in silk fibroin porous microballoon preparation process, fibroin albumen chain is as template pair
Mineral regulate and control so that mineral are regular arrangement crystallization in fibroin albumen template, and macro morphology is for having honeycomb
The porous microsphere structure of shape.Used herein to raw material in containing hydroxyapatite, fibroin albumen and class bone
The compositions such as apatite, natural safety, recycling can be absorbed by organism；There is the self-assembled structures of multilayer, this
Plant and formed based on self assembly in fibroin template by one-dimensional or two-dimentional apatite nano structured unit, at fibroin mould
Plate is outside generates the spherical porous shell that a circle is hard.This fibroin microsphere also has the physico-chemical property of himself uniqueness,
As big in specific surface area, water insoluble etc.；Its electronegative characteristic so that because of electric charge between microballoon and microballoon
Repel and be difficult to reunite, and with the medicine (such as adriamycin) of positively charged, electrostatic adsorption can occur, carry
High microballoon bag loading capability.
Controllable sustained-release effect of the present invention is presented as that cellular loose structure can provide individually for medicine
The absorption of storage area, beneficially high amount of drug, can be with the sustained release efficiency of regulating drug by regulation pH value.
The method technique is simple, processing ease, good biocompatibility.Cannot be only used for silkworm, wild silkworm (tussah silk and
Wild silk yarn etc.) etc. the mineralising of fibroin protein film, even can be used for other biological macromolecule material in order to prepare microballoon.
This invention enriches the type of fibroin albumen microballoon, can as having the carrier of bioactivator, load enzyme,
Nucleic acid, polypeptide, pharmaceutical grade protein etc., be applied to medical diagnosis on disease and treatment etc..Medicament slow release, organizational project,
Enzyme engineering, photocatalysis field etc. have application prospect.
Due to the utilization of technique scheme, the present invention compared with prior art has a feature highlighted below:
(1) excellent biocompatibility；The fibroin albumen and the apatite that use in raw material are all natural active matter,
It is commonly used to biological medicine industry, to cell or tissue non-toxic reaction；Cell experiment shows higher thin
Born of the same parents' adhesion rate and the rate of increase, have good biocompatibility, and biological safety is high, can meet in biomedicine
Application；
(2) simple to operate, with short production cycle: extraction process is simple, efficient, there is no complicated loaded down with trivial details preparation
Journey；
(3) not using organic or toxic reagent, environment and testing crew are not damaged by preparation process；
(4) surface has regular shape uniform honeycomb loose structure, is loaded on by model drug (adriamycin)
The carried medicine sustained-release microsphere of preparation in microballoon, compared with ganoid fibroin microsphere, it is possible to significantly improve pattern medicine
The drugloading rate of thing, and can make medicine slowly, linearly discharge；
(5) there is pH sensitiveness, it is possible to discharged from microballoon by the change regulating drug of solution ph
Speed, has controllability；
(6) prepare there is rigid shell electronegative fibroin albumen microballoon, it is to avoid pure silk fibroin microballoon
Between mutual bonding and problem unstable in aqueous, microspherulite diameter is evenly distributed, and improves the life of medicine
Thing availability.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscopic picture of cellular silk fibroin porous microsphere drug slow-released carrier in embodiment 1.
Fig. 2 is that in embodiment 2, cellular silk fibroin porous microsphere drug slow-released carrier bag carries after adriamycin medicine
Slow release effect figure in different pH solution.
Fig. 3 is that in embodiment 3, cellular silk fibroin porous microsphere drug slow-released carrier bag carries after adriamycin medicine
Join Bcap-37 tumour cell, the cell morphology figure after different time processes.
Detailed description of the invention
Below by embodiment, the present invention is described in further detail, and following example are the solutions to the present invention
Release and the invention is not limited in following example.
Embodiments of the invention are as follows:
Embodiment 1
In the present embodiment, the preparation method of cellular silk fibroin porous microsphere drug slow-released carrier includes as follows successively
Step:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain silk
The fibroin aqueous solution；
(2) the silk fibroin protein solution concentration in step (1) is adjusted to 0.1mg/mL and ultrasonic disperses,
Loading and be placed in the human body simulation body fluid of different multiples in bag filter soaking, solution temperature is 37 DEG C, rotating speed:
100 revs/min.
(3) shaking table processes the time is 14 days, and the simulated body fluid in every 24 hours exchonge step (2) obtains
Microballoon suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing for several times, centrifugal point
From, freeze-drying 5 hours, it is thus achieved that before the apatite of 1～5 μm/fibroin albumen microballoon；
(7) human mesenchymal stem cells's biocompatibility experiment shows, the silk fibroin porous microballoon tool of non-medicine carrying
There is good biocompatibility.
Embodiment 2
The mineralization method promoting the tussah silk peptide of cell growth in the present embodiment in turn includes the following steps:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain silk
The fibroin aqueous solution；
(2) the silk fibroin protein solution concentration in step (1) is adjusted to 50mg/mL and ultrasonic disperses,
Loading and be placed in the human body simulation body fluid of different multiples in bag filter soaking, solution temperature is 37 DEG C, rotating speed:
200 revs/min.
(3) shaking table processes the time is 1 day, and the simulated body fluid in every 24 hours exchonge step (2) obtains micro-
Ball suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing for several times, centrifugal point
From, it is dried 10 hours at 60 DEG C, it is thus achieved that the apatite of 0.5～2 μm/fibroin albumen microballoon；
(5) it is 1:100 by the mass ratio of ADMh Yu fibroin albumen microballoon, by ADMh solution
Being well-dispersed in fibroin albumen microspheres solution, shake on shaking table, 37 DEG C of temperature are bathed 12 hours, obtain mixed liquor.
(6) by the mixed liquor in step (5), supernatants are removed through 7000 revs/min of centrifugal treating, 50%
Ethanol wash centrifugal segregation supernatant, freeze-drying again, obtain bag and carry the fibroin albumen sustained-release micro-spheres of adriamycin.
(7) the fibroin albumen sustained-release micro-spheres that the bag in step (6) carries adriamycin is used for being sustained experiment, and pH is
When 7.4, the fibroin albumen sustained-release micro-spheres of bag load adriamycin is 38.5% the burst size of 60 hours, and pH is
When 6.2, the fibroin albumen sustained-release micro-spheres of bag load adriamycin is 84.2% the burst size of 24 hours.
Bag carry the fibroin albumen sustained-release micro-spheres of adriamycin slow release effect in different pH solution as in figure 2 it is shown,
Experiment shows: (normal body fluid environment), the rate of release of 120 hours interior adriamycins when pH value is 7.4
The least, within 100 hours, burst size is about 46.3%；And when pH value is 6.2 (with tumor tissue cell's epimatrix
Environment is close), within 24 hours, i.e. discharge 84.2%, rate of release is clearly.Visible, the embodiment of the present invention will
Model drug (adriamycin) bag is loaded in the drug bearing microsphere prepared by fibroin albumen base microballoon can controllably (be passed through
Regulation pH) slowly discharge adriamycin.The most provable fibroin albumen microballoon having embedded adriamycin has significantly
Sustained release property, and there is pH sensitiveness.
Embodiment 3
The mineralization method promoting the tussah silk peptide of cell growth in the present embodiment in turn includes the following steps:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain toothed oak
The fibroin protein aqueous solution；
(2) the tussah silk fibroin solution concentration in step (1) is adjusted to 2mg/mL and ultrasonic carries out point
Dissipating, load and be placed in the human body simulation body fluid of 5 times in bag filter soaking, solution temperature is 37 DEG C, rotating speed:
20 revs/min.
(3) shaking table processes the time is 7 days, and the simulated body fluid in every 24 hours exchonge step (2) obtains micro-
Ball suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing for several times, centrifugal point
From, freeze-drying 48 hours, it is thus achieved that the apatite/Wild antheraea pernyi silk fibroin microsphere of 1～5 μm；
(5) collecting logarithmic phase tumour cell, every hole adds 200 μ L, bed board cell adjust density to 10000/
Hole.At the bottom of cell monolayer is paved with hole, add the microballoon being loaded with DOX medicine of gradient concentration, continue cultivation 4
Hour, 12 hours, 48 hours.Experimental result shows, drug bearing microsphere 4 hours and 12 hours is to tumour cell
Toxic and side effect is the most weak, can kill most cells at 48 hours.
The cellular silk fibroin porous microsphere drug slow-released carrier bag of embodiment joins after carrying adriamycin medicine
Bcap-37 tumour cell, cell morphology after different time processes is as it is shown on figure 3, the most provable logical
Cross the present invention and can significantly reduce drug bearing microsphere to Normocellular toxic and side effect, and discharge medicine at carcinoma cells
Kill cancer cell.
Embodiment 4
The mineralization method promoting the tussah silk peptide of cell growth in the present embodiment in turn includes the following steps:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain silk
The fibroin aqueous solution；
(2) the silk fibroin protein solution concentration in step (1) is adjusted to 1mg/mL and ultrasonic disperses,
Loading and be placed in bag filter in the human body simulation body fluid after modifying soaking, solution temperature is 37 DEG C, rotating speed: 66
Rev/min.
(3) time that the system in step (2) processed is 5 days, obtains microballoon suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing for several times, centrifugal point
From, freeze-drying 48 hours, it is thus achieved that the apatite of 1～5 μm/fibroin albumen microballoon；
(5) it is 50:100 by the mass ratio of ADMh Yu fibroin albumen microballoon, by ADMh solution
Being well-dispersed in fibroin albumen microspheres solution, shake on shaking table, 37 DEG C of temperature are bathed 24 hours, obtain mixed liquor.
(6) by the mixed liquor in step (5), supernatants are removed through 7000 revs/min of centrifugal treating, 75%
Ethanol wash centrifugal segregation supernatant, freeze-drying again, obtain bag and carry the fibroin albumen sustained-release micro-spheres of adriamycin.
By the provable microballoon of the present invention of the embodiment of the present invention, there is obvious sustained release property, and there is pH sensitiveness,
Drug bearing microsphere can be significantly reduced to Normocellular toxic and side effect by the present invention.Therefore the present invention is that a kind of bag carries
Drug efficiency is high, the significant slow releasing carrier of medication of slow release effect.
Finally, in addition it is also necessary to it is noted that listed above be only the present invention be embodied as example.Obviously, originally
Invention is not limited to above example, it is also possible to have many deformation.Those of ordinary skill in the art can be from this
All deformation that bright disclosure directly derives or associates, are all considered as protection scope of the present invention.
Claims (6)
1. a preparation method for cellular silk fibroin porous microsphere drug slow-released carrier, includes walking as follows successively
Rapid:
(1) the fibrous fibroin formed after degumming silkworm cocoons is dissolved, after filtering, dialyse, concentrating, obtain silk
The fibroin aqueous solution；
(2) the silk fibroin protein solution concentration in step (1) is adjusted to 0.1～50mg/mL and ultrasonic carries out
Dispersion, loads in the human simulation body fluid being placed in different times concentration in bag filter and soaks, and solution temperature is 37 DEG C,
At the uniform velocity shaking table processes；
(3) shaking table processes the time is 1-14 days, the human simulation body fluid in every 24 hours exchonge step (2),
Obtain microballoon suspension；
(4) by the microballoon suspension deionized water in step (3) and absolute ethanol washing for several times, centrifugal point
It is dried 5-48 hour at, freeze-drying or 60 DEG C, it is thus achieved that particle diameter is the apatite/fibroin albumen of 0.2～5 μm
Microsphere powder, as slow releasing carrier of medication.
The preparation of a kind of cellular silk fibroin porous microsphere drug slow-released carrier the most according to claim 1
Method, it is characterised in that: described step (4) middle deionized water and absolute ethanol washing 3～5 times.
The preparation of a kind of cellular silk fibroin porous microsphere drug slow-released carrier the most according to claim 1
Method, it is characterised in that: the fibroin raw material in described step (1) uses bombyx mori silk fibroin, silk gland protein, wild silkworm
Fibroin, spider silk fibroin or recombinant fibroin, but it is not limited to this.
The preparation of a kind of cellular silk fibroin porous microsphere drug slow-released carrier the most according to claim 1
Method, it is characterised in that: one times of concentration of described human simulation body fluid uses mode to prepare: 800ml go from
Sub-water adds NaCl:7.996g, NaHCO in order3: 0.350g, KCl:0.224g,
K2HPO43H2O:0.228g, MgCl2·6H2O:0.305g, CaCl2: 0.278g, Na2SO4:
0.071g, then with the trishydroxymethylaminomethane/hydrochloride buffer of 50mmol/L, the pH value of simulated body fluid is adjusted
It is 7.40, is settled to 1000ml.
The preparation of a kind of cellular silk fibroin porous microsphere drug slow-released carrier the most according to claim 1
Method, it is characterised in that: described size of pharmaceutical particles is less than the honeycomb structure in apatite/fibroin albumen microballoon
Footpath size.
The preparation of a kind of cellular silk fibroin porous microsphere drug slow-released carrier the most according to claim 1
Method, it is characterised in that: described medicine includes the cancer therapy drugs such as ADMh but is not limited to cancer therapy drug,
The bag that can also be used for other medicines carries.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106637969A (en) * | 2016-09-21 | 2017-05-10 | 东莞市联洲知识产权运营管理有限公司 | Tussah fibroin protein microsphere including three-dimensional porous structure and production method thereof |
| CN107050511A (en) * | 2017-04-11 | 2017-08-18 | 江南大学 | A kind of biological enzyme fibroin albumen/calcium phosphate composite materials preparation method |
| CN107184564A (en) * | 2017-05-17 | 2017-09-22 | 浙江大学 | A kind of method of the nuclear shell structure nano microballoons of synthesis fibroin albumen@ZIF 8 |
| CN109876147A (en) * | 2019-02-28 | 2019-06-14 | 李琳 | A kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier |
| CN110251480A (en) * | 2019-06-26 | 2019-09-20 | 浙江大学 | A kind of fibroin albumen of core-shell structure/manganese dioxide complex microsphere pharmaceutical carrier and preparation method |
| CN110538164A (en) * | 2019-10-09 | 2019-12-06 | 安徽中医药大学 | PH-sensitive hydroxyapatite/zein nano-drug carrier and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526801A (en) * | 2012-02-20 | 2012-07-04 | 浙江大学 | Preparation method for flexible sericin protein/hydroxyapatite composite membrane |
| CN103965310A (en) * | 2014-04-21 | 2014-08-06 | 浙江大学 | Self-assembling method for preparing fibroin microspheres |
| CN104825398A (en) * | 2015-04-24 | 2015-08-12 | 中原工学院 | Preparation method and use of surface mesoporous silk fibroin microsphere drug slow-release carrier |
-
2016
- 2016-05-27 CN CN201610369157.0A patent/CN105832682B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102526801A (en) * | 2012-02-20 | 2012-07-04 | 浙江大学 | Preparation method for flexible sericin protein/hydroxyapatite composite membrane |
| CN103965310A (en) * | 2014-04-21 | 2014-08-06 | 浙江大学 | Self-assembling method for preparing fibroin microspheres |
| CN104825398A (en) * | 2015-04-24 | 2015-08-12 | 中原工学院 | Preparation method and use of surface mesoporous silk fibroin microsphere drug slow-release carrier |
Non-Patent Citations (1)
| Title |
|---|
| 蒋伏广: "再生丝素蛋白/羟基磷灰石多孔复合材料的制备", 《化学世界》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106637969A (en) * | 2016-09-21 | 2017-05-10 | 东莞市联洲知识产权运营管理有限公司 | Tussah fibroin protein microsphere including three-dimensional porous structure and production method thereof |
| CN106637969B (en) * | 2016-09-21 | 2020-01-10 | 上海寓尚电子商务有限公司 | Tussah silk fibroin microsphere containing three-dimensional pore structure and preparation method thereof |
| CN107050511A (en) * | 2017-04-11 | 2017-08-18 | 江南大学 | A kind of biological enzyme fibroin albumen/calcium phosphate composite materials preparation method |
| CN107184564A (en) * | 2017-05-17 | 2017-09-22 | 浙江大学 | A kind of method of the nuclear shell structure nano microballoons of synthesis fibroin albumen@ZIF 8 |
| CN107184564B (en) * | 2017-05-17 | 2020-06-16 | 浙江大学 | Method for synthesizing silk fibroin @ ZIF-8 core-shell structure nano microspheres |
| CN109876147A (en) * | 2019-02-28 | 2019-06-14 | 李琳 | A kind of preparation method and applications of fibroin albumen microsphere drug slow-released carrier |
| CN110251480A (en) * | 2019-06-26 | 2019-09-20 | 浙江大学 | A kind of fibroin albumen of core-shell structure/manganese dioxide complex microsphere pharmaceutical carrier and preparation method |
| CN110251480B (en) * | 2019-06-26 | 2020-05-12 | 浙江大学 | Silk fibroin/manganese dioxide composite microsphere drug carrier with core-shell structure and preparation method thereof |
| CN110538164A (en) * | 2019-10-09 | 2019-12-06 | 安徽中医药大学 | PH-sensitive hydroxyapatite/zein nano-drug carrier and application thereof |
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